Aminoacyl-tRNA synthetases are essential enzymes that catalyze the addition of amino acids to their cognate tRNAs as a first step in protein synthesis. These essential enzymes are separated into two classes based on the presence of unique sequence motifs and overall structure of their catalytic domains. Class I synthetases have two highly conserved amino acid motifs, i.e., the sequences HIGH (SEQ ID NO: 1) and KMSKS (SEQ ID NO: 2), within the catalytic domains of its ten members. In contrast, the Class II synthetases have three highly degenerate sequence motifs, referred to as motifs 1-3. Over the past twenty years, several additional roles for tRNA synthetases were discovered, including RNA splicing, nuclear export and regulation of gene transcription. These additional functions have been acquired during the long evolution of these ancient enzymes. Many of these added functions are a result of appended domains, which have been fused to the core synthetase sequences. In higher eucaryotes, the appended domains of two synthetases have been demonstrated to have functional biological roles unrelated to the function of the core enzyme. For instance, fragments of two human tRNA synthetases, i.e., tyrosyl-tRNA synthetase (TyrRS) and tryptophanyl-tRNA synthetase (TrpRS), have been demonstrated to have cytokine-like activities. Two related fragments of human TrpRS, known as mini-TrpRS and T2-TrpRS, are negative regulators of angiogenesis. Human TyrRS (SEQ ID NO: 3) can be readily separated into two active fragments. A C-terminal appended domain fragment has activity similar to the pro-inflammatory cytokine endothelial-monocyte-activating polypeptide II (EMAP II), while an N-terminal fragment (mini-TyrRS, residues 1-364 of SEQ ID NO: 3) induces angiogenesis.
Angiogenesis is a tightly regulated process in which a careful balance between pro-angiogenic and anti-angiogenic factors must be maintained. Disruption of this balance, leading to excessive or insufficient growth of blood vessels, is associated with diseases such as age-related macular degeneration, rheumatoid arthritis, delayed wound healing, as well as many other conditions. Regulation is controlled through a variety of processes including transcriptional and translational control, post-translational modifications and processing of the ligand. Other proangiogenic cytokines, including tumor necrosis factor-alpha and hepatocyte growth factor, are generated by proteolytic cleavage of precursor proteins. Similarly, cleavage by proteases releases active cytokine fragments from human TrpRS and TyrRS.
The TrpRS and TyrRS enzymes of higher eukaryotes are composed of a core catalytic region that includes a Rossmann fold having a number of alpha coils interspersed with beta sheet segments. The Rossmann fold catalytic domain of human TyrRS (residues 1 through 230 of SEQ ID NO: 3) includes a hydrogen bond tether between the α5 coil of the Rossmann fold domain and the α14 coil of the anticodon recognition domain (see FIG. 3, Bottom Panel, and FIG. 4, Top Panel). The tether partially blocks the Glu-Leu-Arg (ELR) motif in the α5 coil of the active site domain of the protein.
The catalytic domain of human TyrRS and TrpRS are each homologous to the catalytic domains of their respective corresponding bacterial and lower eukaryotic enzymes, appended with a C-terminal or N-terminal extension, respectively. The C-terminal extension of human TyrRS shares about 51% sequence identity to the pro-inflammatory cytokine EMAP II. In each case, the full-length enzymes are inactive as cytokines, though functional as synthetases. When the extensions unique to higher eukaryotes are removed, the enzymes become active cytokines capable of controlling angiogenesis.
It has now been discovered that opening the separation between the α5 coil of the catalytic Rossmann fold domain and the α14 coil of the anticodon recognition domain relative to the separation of these coils in native human TyrRS renders the protein angiogenic. The present invention provides TyrRS protein variants and fragments thereof, which are useful for stimulating angiogenesis in mammalian tissues.